1. Field of the Invention
This invention relates to applications of DNA fingerprinting and the use of DNA markers in a number of different fields including, but not limited to, plant and animal breeding variety or cultivar identification, diagnostic medicine, disease diagnosis in animals and plants, identification of genetically inherited diseases in humans, family relationship analysis, forensic analysis, and microbial typing.
More specifically, this invention relates to methods for DNA fingerprinting and for detecting specific DNA markers in genomes ranging from microorganisms to higher plants, animals and humans. The invention also relates to synthetic DNA molecules and products based thereon which are used in the methods of the invention in the different fields of application.
2. Description of Related Art
1. DNA Fingerprinting
DNA fingerprinting or DNA typing, as well as other methods of genotyping, profiling and DNA identification analysis, refer to the characterization of either similarities or one or more distinctive features in the genetic make up or genome of an individual, a variety or race, or a species. The general rule is that the closer the genetic relationship is, the greater the identity or more appropriate the similarity of genomes, and consequently distinctive features in the genome will be rarer. These similar or distinctive features can be revealed by analyzing the DNA of an organism after cleaving the DNA with a restriction endonuclease. Restriction endonucleases are enzymes which recognize short nucleotide sequences, usually 4 to 8 bases in length and cleave the two DNA strands, thereby producing fragments of DNA of discrete length. Because of their high degree of sequence specificity, restriction endonucleases will cleave DNA molecules in a very specific fashion. The result is that a reproducible set of DNA fragments will be produced. DNA fragments can be fractionated according to their length on porous matrices, or gels, yielding typical banding patterns, which constitutes a DNA fingerprint of the organism's genetic makeup.
2. DNA polymorphisms
When the fingerprints of very closely related species, varieties or races are compared, the DNA fingerprints can be identical or very similar. When differences are observed within otherwise identical DNA fingerprints, such differences are referred to as DNA polymorphisms: these are new DNA fragments which appear in a fingerprint. The DNA is said to be polymorphic at that position and the novel DNA fragment can be used as a DNA marker. DNA polymorphisms detected in DNA fingerprints obtained by restriction enzyme cleavage can result from any of the following alterations in the DNA sequence: mutations abolishing the restriction endonuclease target site, mutations creating new target sites, insertions, deletions or inversions between the two restriction sites.
Such DNA polymorphisms are generally referred to as RFLP, Restriction Fragment Length Polymorphisms. Such mutational changes will behave as bona fide genetic markers when they are inherited in a mendelian fashion. Consequently, DNA polymorphisms can be used as genetic markers in much the same way as other genetic markers: in parentage analysis, in genetic studies on the inheritance of traits, or in the identification of individuals.
3. DNA fingerprinting techniques
For almost all living organisms, except virusses, restriction digests of the total genomic DNA of the organisms yield so many bands that it is not possible to score individual bands. Therefore, all methods for DNA fingerpinting are based on the principle that only a small fraction of the DNA fragments are visualized so as to yield a simple banding pattern which constitutes the DNA fingerprint.
The most widely utilized method involves digesting the DNA of the organism with restriction endonucleases, fractionating the restriction fragments by gel electrophoresis, transferring and binding the fractionated DNA fragments onto membranes and hybridizing the membrane with a specific DNA fragment ("probe"). The DNA fragment will form double-stranded DNA molecules with the DNA fragment (or fragments) on the membrane which has (have) complementary nucleotide sequences. When the probe is tagged with a visualizable marker, the DNA fragment to which the probe is attached can be visualized. The procedure is generally referred to as "Southern hybridization". When differences are observed in the sizes of the corresponding restriction fragments to which the probe attaches in closely related genomic DNA molecules, these differences are referred to as DNA polymorphisms, more specifically restriction fragment length polymorphisms. The restriction fragment length differences correspond to the different allelic forms of the genetic locus recognized by the DNA probe. Although the Southern hybridization method for DNA fingerprinting has been widely used, the method is laborious and time consuming.
Furthermore, the method has a low resolution and can thus only be used to score single loci or a few loci at most in a single reaction.
4. Polymerase chain reaction
The Polymerase Chain Reaction (PCR) technique is a method for synthesizing specific DNA fragments in vitro. The method relies on the use of specific oligonucleotides which will attach to unique sequences on a DNA molecule and a thermostable DNA polymerase. The oligonucleotides are designed in such a way that they can anneal to the opposite strands of the DNA and serve as primers in a DNA synthesis reaction in such a way that each will direct the synthesis of new DNA strands. Hence, in one round of synthesis a complete copy of the DNA molecule between the primers will be made, so that the DNA between the primers is duplicated. Each round of DNA synthesis results in the doubling of the amount of DNA, hence leading to the amplification of the DNA comprised between the two primers. Consequently, the PCR technique allows one to synthesize a precise DNA segment using a small amount of "substrate DNA".